The p53 tumor suppressor plays a key role in maintaining genomic integrity and preventing tumorigenesis. MDM2, a RING-finger ubiquitin E3 ligase, plays a major role in the regulation of p53 levels. MDM2 ubiquitinates p53 and targets it for proteasome-mediated degradation. MDMX, an MDM2 homolog, also plays an indispensible role in controlling p53. MDMX assists MDM2 in suppressing p53 activity and levels, while MDM2 mediates MDMX ubiquitination and degradation in response to DNA damage. As MDM2, and possibly MDMX as well, can be transcription ally induced by p53, they form a p53-MDM2-MDMX feedback loop. This loop ensues a normal homeostasis of these proteins in cells. Recent studies have also shown that the p53-MDM2-MDMX loop is regulated via deubiquitination by counteraction of several deubiquitinating enzymes (DUBs) from the ubiquitin specific protease (USP) family. USP7 deubiquitinates and stabilizes all the three players, whereas USP10 deubiquitinates p53 only and USP2a deubiquitinates MDM2 and MDMX, providing another layer of precise and dynamic regulation of the p53 pathway. However, whether this loop is regulated by DUBs other than USP family is not known. We recently discovered that an OTU-domain containing protease (OTU) family member, Otubain 1 (Otub1), as a novel regulator of the p53-MDM2-MDMX loop. We show that over expression of Otub1 reverses MDM2-mediated p53 ubiquitination, stabilize p53 in cells, and drastically induce p53- dependent apoptosis and cell growth inhibition. Over expression of a dominant-negative mutant of Otub1 or ablation of endogenous Otub1 by siRNA significantly attenuated p53 activation in response to DNA damage. These results suggest a crucial role for Otub1 in the regulation of p53 signaling. To further gain insight into the regulation, we will investigate the molecular and biochemical mechanisms underlying the role of Otub1 in regulating the p53 pathway in Aim 1. As Otub1 also suppresses MDMX ubiquitination and stabilizes MDMX in cells and, intriguingly, the stabilized MDMX by Otub1 is highly phosphorylated, we will further characterize the mechanism underlying Otub1 regulation of MDMX as well as its significance in Aim 2. Finally, as Otub1 drastically induces p53-dependent cell growth inhibition in cell culture system, we will examine if Otub1 suppresses tumor growth in vivo using mouse xenograft and knockout models in Aim 3. We will examine whether Otub1 expression is deregulated and/or the Otub1 gene is mutated in human cancers. We will also examine the mechanism underlying the Otub1 regulation of p53 signaling in response to DNA damage. Completion of these aims would not only further our understanding of how p53 function is properly regulated through dynamic ubiquitination and deubiquitination and how deregulation of this dynamics contributes to tumorigenesis, but also aid the search for novel cancer therapeutics to re-activate p53 in wild-type p53 containing cancers.